Abstract 2517: Optimizing breast cancer therapy by inhibiting the adenosine receptor and oxygen consumption
Authors: Sadna Budhu, Mayuresh Mane, Mamadou A. Bah, Juan Zurita, Inna Serganova, Soe Min, Anais Assouvie, Jedd D. Wolchok, Jason Koutcher, Vladimir Ponomarev, Taha Merghoub
Journal: Cancer Research (2023)

Abstract Immune checkpoint blockade has shown remarkable promise in melanoma and other tumor types. However, a large proportion of patients do not respond or develop acquired resistance. Tumors can activate multiple checkpoints and immunosuppressive pathways to evade immune surveillance, which are likely a cause of treatment failure. Therefore, inhibition of multiple checkpoints may be necessary for maximal efficacy of immunotherapies. The adenosine A2 receptor (A2aR), was shown to function as an immune checkpoint. Its blockade inhibited tumor growth and metastases and synergized with other checkpoint inhibitors. We propose that using AZD4635, a potent and selective A2aR antagonist, will enhance the therapeutic efficacy of anti-PD-1/PD-L1. In addition, we propose that blocking tumor oxygen consumption using deferiprone (DFP), phenformin (Phen) and metformin (Met) will further enhance A2aR and PD-1/PD-L1 blockade efficacy. We evaluated the effects of MET, Phen, DFP, and AZD4635 on tumor cells, T cell, and macrophage proliferation and effector function in vitro. IC50 measurements showed that T cells are the most sensitive to inhibition by these drugs, while 4T1 tumor cells appear to be the least sensitive. Naïve mouse T cells were activated with anti-CD3 and anti-CD28 coated dynabeads in the presence of titrating doses of all four drugs for 72 hours then analyzed by flow cytometry for proliferation and activation status. AZD4635 and DFP increased the expression of the activation marker CD25 (IL2Ra) on CD8 T cells. In addition, AZD4635 also increased expression of Granzyme B on both CD4 and CD8 T cells. Met appears to have little to no effect on T cell proliferation activation. We utilized the Seahorse XF Mito Stress Test assay to measure the mitochondrial respiratory activity of T cells in the presence these drugs. T cells were activated with dynabeads for 72 hours and then incubated with titrating doses of the drugs overnight before running on the Seahorse assay. DFP and Phen had the strongest effect on Maximal Respiration and Spare Respiratory Capacity at dilutions of 1uM followed by 16uM. A similar, albeit less profound effect was observed in groups treated with Met and AZD4635. Lastly, we obtained and generated two mouse breast cancer cell lines (4T1 and E0771) bearing HIF-1 reporter systems. We treated 4T1-HRE and E0771 cells with increasing doses of cobalt chloride (CoCl2) to chemically mimic hypoxia by inducing HIF1a expression. Both 4T1-HRE and E0771-HRE demonstrated increased luciferase activity that correlated with increasing doses of CoCl2 in vitro. Future experiments will focus on characterizing the adenosine pathway in vivo in 4T1-HRE and E0771-HRE tumors and examine how drugs that target the adenosine A2AaR receptor (AZD4635), and oxygen consumption (DFP, Phen and Met) influence tumor oxygen consumption in vivo as well as the activation states of immune cells in the tumor microenvironment. Citation Format: Sadna Budhu, Mayuresh Mane, Mamadou A. Bah, Juan Zurita, Inna Serganova, Soe Min, Anais Assouvie, Jedd D. Wolchok, Jason Koutcher, Vladimir Ponomarev, Taha Merghoub. Optimizing breast cancer therapy by inhibiting the adenosine receptor and oxygen consumption [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2517.